Mycobacterium intracellulare induces a Th17 immune response via M1-like macrophage polarization in canine peripheral blood mononuclear cells

Mycobacterium avium-intracellulare complex (MAC) is one of the most prevalent pathogenic nontuberculous mycobacteria that cause chronic pulmonary disease. The prevalence of MAC infection has been rising globally in a wide range of hosts, including companion animals. MAC infection has been reported in dogs; however, little is known about interaction between MAC and dogs, especially in immune response. In this study, we investigated the host immune response driven by M. intracellulare using the co-culture system of canine T helper cells and autologous monocyte-derived macrophages (MDMs). Transcriptomic analysis revealed that canine MDMs differentiated into M1-like macrophages after M. intracellulare infection and the macrophages secreted molecules that induced Th1/Th17 cell polarization. Furthermore, canine lymphocytes co-cultured with M. intracellulare-infected macrophages induced the adaptive Th17 responses after 5 days. Taken together, our results indicate that M. intracellulare elicits a Th17 response through macrophage activation in this system. Those findings might help the understanding of the canine immune response to MAC infection and diminishing the potential zoonotic risk in One Health aspect.

www.nature.com/scientificreports/ produce anti-inflammatory cytokines and cause persistent infection 8,9 . Macrophages have been reported to exhibit transformation from the M1 to the M2 phenotype over the course of mycobacterial infection 7,10 . However, recent studies have shown that Mycobacterium-infected macrophages polarize to a unique macrophage population that is clearly distinguishable from the M1 and M2 macrophage subsets 11 . In particular, M. intracellulareinfected macrophages represent a novel polarized population that downregulates Th1/Th2 cell production while driving Th17 polarization 12 .
In mycobacterial infection, CD4 + T cells are the most dominant in the immune response 13 . The Th1/Th17 balance is thought to be important for protection against mycobacteria 14,15 . The Th1 immune response is essential for controlling mycobacterial infection. Th17 cells participate in the antimycobacterial response by accelerating the accumulation of Th1 cells at an early stage 16,17 . However, the Th17 response has pathological effects during MAC infection under Th1-diminished conditions. Th17 development induces excessive neutrophilic pulmonary inflammation by reducing Th1 responses and can increase susceptibility to systemic MAC infection 15 . Inflammation and neutrophil recruitment driven by Th17 cells play a pivotal role in granuloma formation and mycobacterial disease development 18,19 .
As mycobacteriosis caused by MAC in dogs has been consistently increased, host immune response should be revealed to understand the pathogenesis in MAC-infected dogs. As the first step, an in vitro canine model was developed to investigate the immune response to M. intracellulare infection. Transcriptomic analysis in canine monocyte-derived macrophages was carried out to identify activated macrophages driven by the bacterial infection. Also, the T cell response was investigated using M. intracellulare-infected autologous macrophages. These results will help further our understanding of the pathogenesis of M. intracellulare-related crosstalk between macrophages and T helper cells. Furthermore, this study helps to establish a basis for research on MAC infections, which occur in a wide range of hosts, as well as for understanding the mechanism of M. intracellulare infection in dogs.

Results
Transcriptome analysis of canine monocyte-derived macrophages against M. intracellulare infection. To identify the canine immune response against M. intracellulare, canine MDMs were analyzed through RNA-Seq after infection with M. intracellulare for 0, 6, 24, and 72 h. Sixteen cDNA libraries from noninfected and M. intracellulare-infected cells were sequenced. A total of 1542 differentially expressed genes (DEGs) were significantly expressed in the M. intracellulare infection group compared to the uninfected group. Transcriptional profiles were analyzed by Ingenuity Pathway Analysis (IPA) using the significant DEGs ( |fold change| ≥ 1.5 ). A comparison analysis was performed using canonical pathways that were expressed at each time point ( Table S1). The pathways were also involved in the induction of the T cell response. The activated pathways TREM1 Signaling, HMGB1 Signaling and Dendritic Cell Maturation were associated with the Th1 response, and other activated pathways (Role of Th17F in Allergic Inflammatory Airway Diseases, IL-6 Signaling, Th17 Activation Pathways, IL-23 Signaling Pathway) were related to the Th17 response. In addition, the inactivated pathways (LXR/RXR Activation, PPARα/RXRα Activation, PPAR Signaling) were involved in the inhibition of the Th17 response. Of the molecules expressed in these pathways, Th1 /Th17 cell response-related molecules (CCL3, CCL4,  CCL5, CCL7, IL1B, IL6, CSF2, CXCL8, ICAM1, S100A8, PTGS2, CD40, CD80) were commonly upregulated during M. intracellulare infection. However, IL15, which is known to be related to the Th1 cell response, was inactivated (Supplementary Table S1).
Canine macrophages differentiate into M1-like macrophages after M. intracellulare infection. After Table S4). The activation status was predicted by the z-score. Figure 1A shows that most M1 macrophage-associated pathways were activated, whereas Fig. 1B shows that M2 macrophage-associated pathways were negatively regulated or inactivated. The grey boxes in Supplementary Table S5 indicate pathways that were continuously activated or inactivated throughout M. intracellulare infection. However, some pathways were not consistent with the M1 activation states. In particular, apoptosis-related pathways (Apoptosis Signaling, Myc Mediated Apoptosis Signaling, Induction of Apoptosis by HIV1, Production of Nitric Oxide and Reactive Oxygen Species in Macrophages) were negatively regulated over time. The Apoptosis Signaling pathway was predicted to be inactivated throughout the infection.
Because of this discrepancy with the characteristics of classical M1 macrophages, macrophage activationrelated genes were quantified by RT-qPCR during 72 h of infection. The expression levels of M1 macrophage markers (IL-23, IL-1β, IL-6, TNF-α, IDO1) were significantly upregulated in M. intracellulare-infected MDMs compared to noninfected MDMs ( Fig. 2A). In contrast, M2 macrophage markers did not show any significant changes during M. intracellulare infection (Fig. 2B). An invasion assay also showed that M. intracellulare replicated in canine MDMs after infection. The amount of intracellular M. intracellulare increased after 5 days of   Table S4). These results were also confirmed by analysis of the expression of particular genes by RT-qPCR. Gene expression profiling revealed that the expression of the IL-23, IL-1β, and IL-6 genes, which induce Th17 cell polarization, was significantly increased compared to that in the control group. Among Th1-related genes (IFN-γ, TNF-α, IL-12), only TNF-α showed significantly increased expression. Th2 (IL-4, IL-13) and Treg (IL-10, TGF-β)-related genes did not show significantly increased expression in canine MDMs infected with M. intracellulare (Fig. 2). ). Gene expression profiling of co-cultured lymphocytes showed that Th17-related genes (IL-1β, IL-17) were significantly upregulated compared to the control (Fig. 4B). In particular, the gene expression of IL-17 was highest among cytokines associated with subsets of CD4 + helper T cells. Other cytokines associated with Th1 (IFN-γ, IL-12), Th2 (IL-4, IL-13), and Tregs (IL-10, TGF-β) were not significantly upregulated except for TNF-α. However, TNF-α was significantly expressed only at 12 h, and the expression level was also very low (fold change = 1.007 ± 0.117) . We then analyzed the supernatants of lymphocytes with ELISA to confirm the production of cytokines (Fig. 5). All cytokines related to subsets of CD4 + helper T cells were secreted upon M. intracellulare infection, except the cytokines related to Th2. Th17-related cytokines (IL-17A, IL-1β) expressed higher than other cytokines produced in Th1 cells (TNF-α, IFN-γ) and Tregs (IL-10). T cell responses according to M. intracellulare were identified at a single-cell level using flow cytometry. To confirm the impact of M. intracellulare infected MDMs, canine lymphocytes were co-cultured with nonautologous MDMs infected with M. intracellulare for five days (Fig. 6). CD4 + T cells in the co-cultured cells were analyzed for the Th17 responses ( Supplementary Fig. S3). The canine CD4 + T cells polarized moderately to IL-17-producing cells by M. intracellulare-infected MDMs independent of T cell receptor recognition (Fig. 6A). Next, lymphocytes were co-cultured with the non-autologous MDMs by replacing the antigenic recognition with the anti-CD3 antibody. The canine CD4 + T cells were highly polarized to IL-17-producing cells by replacing MHC recognition. The IL-17 expression was confirmed in lymphocytes co-cultured with infected MDMs treated with anti-CD3 antibody compared to the isotype control and in lymphocytes co-cultured with noninfected MDMs treated with anti-CD3 antibody (Fig. 6D). The IL-17 expression was confirmed in the other two individual canine lymphocytes by comparing the percentage and mean fluorescence intensity (Fig. 6B,C). For validating the antigenic specificity of T cell response, lymphocytes were co-cultured with autologous MDMs, which were non-infected or infected with M. intracellulare, and treated with LPS (Fig. 7A). The percentage of IL-17A-producing CD4 + T cells specific to M. intracellulare was higher than those of IFN-γ-producing CD4 + T cells (Fig. 7B). The production of IFN-γ from CD4 + T cells was hardly observed in the response by M. intracellulare-infected MDMs (Figs. 6, 7). Of note, IFN-γ produced by CD4 + T cells was confirmed by stimulation www.nature.com/scientificreports/ with lipopolysaccharide ( Supplementary Fig. S4). Overall, M. intracellulare activated macrophages to M1-like phenotype, and M. intracellulare-infected macrophages polarized CD4 + T cells to Th17 cells.

Discussion
Mycobacterium avium complex has been highlighted as an emerging opportunistic intracellular pathogen that is ubiquitous in the environment, often leading to high rates of host-pathogen contact. In the case of dogs, it has been reported that domestic dogs are more susceptible to mycobacteriosis caused by MAC 3 . Therefore, it is important to investigate the MAC infection in dogs not only for treatment and diagnosis but also to prevent potential zoonotic risk. However, the susceptibility and pathogenicity of MAC in dogs have been remained to be revealed because of the difficulty to evaluate the canine immune response in vitro. Therefore, we investigated the canine immune response against M. intracellulare infection by co-culture systems of canine T helper cells and MDMs. Our study showed that susceptibility and inflammatory responses to M. intracellulare in dogs were regulated by Th17 cells driven by M1-like macrophage activation. Transcriptome analysis showed that MDMs were activated to the M1-like macrophage state and secreted cytokines to induce Th1/Th17 cell responses to M. intracellulare infection. Top 20 canonical pathways showed that M1 macrophage-related pathways were activated, while M2 macrophage-related pathways were negatively     20 . These molecules induce protective immune responses upon M1 polarization 21 . ICAM1 is an adhesion molecule whose expression is increased by proinflammatory cytokines in an NF-κB-dependent manner 22 . ICAM1 has been reported to regulate macrophage activation by inhibiting M2 polarization 23 . Proinflammatory cytokines also induce CSF2, which activates the expression of cytokines and chemokines to enhance macrophage differentiation to the M1 state 24,25 . TREM1 increases IL-1β production through M1 macrophages as a key player in protective innate immunity during MAC infection 26,27 . CD40 is a representative marker of M1 macrophages involved in antibody isotype switching and vascular inflammation 28 . Conversely, the M2 macrophage marker CD36 was downregulated during M. intracellulare infection. CD36 regulates the nuclear receptor PPARγ as a class B scavenger receptor for the endocytosis of triacylglycerol-rich lipoprotein particles 29 . PPARγ enhances the M2 macrophage response, and lipid modification and repair are fundamental properties of M2 macrophage function 30 .
After identifying the pattern of the immune response by the top 20 canonical pathways, the significantly expressed pathways were analyzed in relation to macrophage activation. The activation states of these pathways showed that most M1 activation-related pathways were activated and they are known to be involved in proinflammatory and protective immune responses against bacterial invasion. On the other hand, M2 activation pathways were negatively modulated and these pathways are related to modulating the nuclear receptor known to enhance M2 activation 29,30 . However, some of the identified pathways were not consistent with the M1 activation state. Especially, apoptosis-related pathways were negatively regulated during the infection. Nevertheless, these states might help explain the reported properties of MAC after macrophage invasion. MAC members are known to replicate within macrophages by preventing apoptotic cell death induced by extrinsic or intrinsic apoptotic pathways [31][32][33] . Our results also showed that the amount of M. intracellulare was increased after 6 days of invasion. However, further study needs to identify the apoptosis of canine macrophages is inhibited by intracellular M. intracellulare. These results differ from the classic M1 phenotype known to have a bactericidal function but are consistent with the MAC characteristics after macrophage invasion. Gene expression profiling of macrophage polarization markers showed that only M1 macrophage-related genes were significantly expressed. These results reveal that canine MDMs differentiate into M1-like macrophages during M. intracellulare infection, although the activation states of pathways involved in macrophage polarization are distinct from the classic M1 and M2 macrophage phenotypes 8,9 . However, recent studies have reported that macrophages polarize into a unique macrophage population during mycobacterial infection 11 . In particular, Tatano et al. reported that M. intracellulare induces a novel type of macrophage population that downregulates the Th1/Th2 cell response, while driving Th17 polarization 12 .
Our results also indicated that the canine MDMs secreted molecules to induce Th17 polarization in response to M. intracellulare infection. The top 20 canonical pathways showed that the activated pathways were related to the Th1/Th17 responses. Among the upregulated molecules of that pathways, chemokines (CCL3, CCL4, CCL5, CCL7) and cytokines (IL-1B, IL-6, CSF2), were responsible for mediation of the innate immune response in Th1 and Th17 cells during mycobacterial infection [34][35][36] . However, most of the upregulated molecules were associated with inducing pathogenic Th17 cells during mycobacterial infection. In particular, CCL3, CCL4, and CCL5 were reported as pathogenic Th17-signature markers during mycobacterial infection 37,38 . CSF2 (GM-CSF) is also known to contribute to the pathogenicity of Th17 cells 39 . The chemokine CXCL8 and the adhesion molecule www.nature.com/scientificreports/ ICAM1 are directly associated with the Th17 response, which induces the maturation and activation of neutrophils during mycobacterial infection 35,40,41 . In addition, the alarmin S100A8, known to contribute to neutrophil accumulation during chronic mycobacterial infection, stimulates IL-6 to promote Th17 differentiation 42,43 . Moreover, PTGS2 (COX-2), which is involved in PGE2 production after mycobacterial infection, causes a dysfunctional immune response that favors the survival and replication of mycobacteria by inducing the development of Th17 cells 44,45 . In addition, negatively modulated pathways (LXR/RXR Activation, PPARα/RXRα Activation, PPAR Signaling) were also related to the induction of the Th17 cell response. Nuclear receptor peroxisome proliferatoractivated receptors, which primarily regulate these pathways, are known to be major negative regulators of Th17 differentiation 46,47 . These results were also confirmed by both of the 'Th1 Pathway' and 'Th17 Activation Pathway' . Only the Th17 Activation Pathway was activated throughout the infection. In addition, gene expression profiling showed that the genes IL-23, IL-1β, and IL-6, which induce Th17 cell polarization, were significantly upregulated during M. intracellulare infection. In contrast, only TNF-α was significantly expressed among molecules related to Th1 cell polarization.
To identify the Th17 cell response against M. intracellulare infection, gene expression was quantified in canine lymphocytes. The result showed that Th17-related genes were significantly upregulated in lymphocytes, similar to the finding in macrophages. Moreover, ELISA indicated that IL-17A and IL-1β were produced at higher levels than other cytokines. Furthermore, IL-17A-expressing CD4 + T cells were higher expressed in comparison to IFN-γ after M. intracellulare infection. The results showed that canine MDMs infected with M. intracellulare provide an environment that may favor Th17 responses. Our results were similar to those of studies that showed the production of IL-17 after infection with the Mycobacterium avium complex. A study by Kannan et al. showed that M. avium infection induced strong induction of IL-17-producing T cells in mice 48 . In particular, a study by Matsuyama et al. reported that the Th17 response was shown to have pathological effects by increasing the susceptibility to systemic M. avium infection under Th1-diminished conditions 15 . Th17 cell development is also known to lead to excessive neutrophilic pulmonary inflammation, which plays a pivotal role in granuloma formation and mycobacterial disease development 18,19 . These results will help explain why most reported cases in dogs showed granulomatous inflammation after mycobacterial disease progression induced by MAC members 3,[49][50][51][52] .
Due to the increase of mycobacterial diseases caused by MAC members in dogs, the need to elucidate the canine immune response has been increased. In this study, the host immune response to M. intracellulare was investigated using in vitro canine model with autologous macrophages and lymphocytes. Our results suggested that M. intracellulare induces M1-like macrophage polarization that provides a cytokine milieu favoring Th17 response in dogs. This study might open the new horizon in the understanding of MAC infection, especially immune response in dogs and help to control the potential public health risk of zoonosis between dogs and humans. Bacterial strains and cultivation. Mycobacterium intracellulare ATCC13950 was kindly provided by Prof. MK Shin from the College of Medicine, Gyeongsang National University, Republic of Korea. M. intracellulare was cultured on Middlebrook 7H10 agar (BD Biosciences, CA, USA) containing 0.1% casitone (BD Biosciences) and Middlebrook 7H9 broth supplemented with 0.04% casitone after 7 days. The infection amount was standardized by using the cells at an optical density of 0.244 at 660 nm (4.9 × 10 7 CFU/ml). The optical density was measured after vigorous vortexing for 30 s to remove clumps.
Generation of canine monocyte-derived macrophages. Monocytes were positively isolated using anti-CD14 magnetic microbeads (Miltenyi Biotec Inc., Bergisch Gladbach, Germany). The purity of isolated monocytes was above 99% and analyzed using the anti-CD14 antibody (TÜK4; Bio-Rad) by flow cytometry (FACS Canto II, BD Biosciences, San Diego, CA, USA) ( Supplementary Fig. S5) www.nature.com/scientificreports/ with 50 ng/ml recombinant human M-CSF (Peprotech Inc., Rocky Hill, HJ, USA) every 2 days for 5 days 53,54 . The morphology was confirmed as the macrophage after 5 days. The anti-dog CD3 antibody (clone CA17.2A12) effectively activated canine lymphocytes as the anti-human CD3 antibody (clone OKT3) does 55,56 . The monocyte-depleted PBMCs were co-cultured with M. intracellulare-infected non-autologous MDMs with or without anti-CD3 antibodies (clone CA17.2A12). The monocytedepleted PBMCs were also co-cultured with non-autologous MDMs with the anti-CD3 antibody. After 5 days of co-cultures, the lymphocytes were restimulated with anti-CD3 antibodies. The activated lymphocytes were analyzed by surface staining and intracellular cytokine staining assay. The data were visualized using FlowJo v10 software (BD Biosciences; https:// www. flowjo. com/). RNA expression, library preparation, and sequencing. Total RNA was isolated from canine MDMs after M. intracellulare infection for 0, 6, 24, and 72 h using an RNeasy Mini Kit (Qiagen, Hilden, Germany). Noninfected MDMs were used as a negative control. Eighty RNA samples were extracted from ten autologous MDMs of both infection and non-infection groups at four-time points. Each five RNA samples were pooled and a total of 16 cDNA libraries was sequenced. The RNA quality was identified by an Agilent 2100 bioanalyzer using an RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and RNA was quantified on an ND-2000 spectrophotometer (Thermo Inc., Waltham, MA, USA). The RNA library was constructed with a QuantSeq 3′ mRNA-Seq Library Prep Kit (Lexogen, Inc., Vienna, Austria) according to the manufacturer's instructions. In brief, the 5′ end of an Illumina-compatible sequence containing an oligo-dT primer was hybridized to the total RNA. After reverse transcription, the RNA template was degraded to synthesize the second strand using an Illumina-compatible linker sequence containing a random primer. To remove all reaction components, the double-stranded library was purified by magnetic beads. The library was amplified with complete adapter sequences and purified from PCR components. High-throughput sequencing was performed with single-end 75-bp sequencing using a NextSeq 500 (Illumina Inc., CA, USA). The RNA-Seq data were deposited into the NCBI Gene Expression Omnibus (GEO) database under accession number GSE158425.

Co-culture of canine lymphocytes and autologous/non-autologous monocyte-derived macrophages infected with
RNA-sequencing read alignment and data analysis. QuantSeq 3′ mRNA-Seq reads were mapped to canFam 3 using Bowtie2 57 . Both the genome assembly sequence and the representative transcript sequences generated Bowtie2 indices. After aligning the transcriptome, transcripts were assembled to identify differentially expressed genes. DEGs were identified based on counts from unique and multiple alignments determined by Bedtools 58 . The read counts were processed based on the quantile normalization method using EdgeR within the R package with Bioconductor 59 . The DEGs were analyzed by Ingenuity Pathway Analysis (IPA; Qiagen Inc., https:// www. qiage nbioi nform atics. com/ produ cts/ ingen uityp athway-analy sis) 60 . Each group was subjected to core and comparative analyzes, and signalling pathways were predicted by the molecule activity predictor in IPA.
Quantification of gene expression. Total RNA was extracted from both canine MDMs infected with M.
intracellulare for 0-72 h and lymphocytes co-cultured with M. intracellulare-infected MDMs for 0-144 h using an RNeasy Mini Kit (Qiagen, Hilden, Germany). Noninfected MDMs and lymphocytes were used as controls. Gene expression was quantified by RT-qPCR to identify the activation of macrophages and T lymphocytes. In addition, RT-qPCR was performed with randomly selected genes to validate the RNA-Seq data ( Supplementary  Fig. S6). cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), and RT-qPCR was performed with Real-Time qPCR 2 × Master Mix (SYBR Green, Elpisbiotech, Daejeon, Republic of Korea) on a Rotor-Gene Q real-time PCR cycler (Qiagen, Hilden, Germany). cDNA was amplified using the canine primers shown in Supplementary Table S6. The amplification conditions were as follows: 95 °C for 3 min and 45 cycles of 95 °C for 3 s and 30 s at 60 °C. A melting curve was constructed for the detection of nonspecific product formation, and the gene expression levels were determined via the 2 −ΔΔCt method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene. The fold change was determined based on the relative gene expression level compared to the control.

Quantification of cytokines.
Lymphocytes were co-cultured at 1 × 10 7 cells/well with M. intracellulareinfected MDMs in 6-well plates for 0-144 h. The cytokine concentrations in the supernatants were determined by ELISA. Canine IL-17A, IL-1β, IL-12, and IL-4 were detected by DuoSet (R&D Systems, Minneapolis, MN, USA) and IFN-γ, IL-6, IL-10, and TNF-α were detected by Quantikine ELISA kits (R&D Systems) according to the manufacturer's instructions. www.nature.com/scientificreports/ Intracellular cytokine staining assay. Lymphocytes were co-cultured with M. intracellulare-infected MDMs to analyze T helper cell polarization. After 5 days of incubation, the cells were incubated with 2 µg/ ml brefeldin A (Sigma) for 4 h. The cells were stained with the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen) and subsequently stained with FITC-anti-CD3 (clone CA17.2A12) and RPE-anti-CD4 (clone YKIX302.9) (both from Bio-Rad Laboratories; Hercules, CA, USA) for 20 min at RT in the dark. After surface staining, the cells were washed with PBS containing 2% FBS and fixed and permeabilized (BD Biosciences, San Diego, CA, USA) for 30 min at 4 °C. The cells were washed twice with BD Perm/Wash buffer (BD Biosciences) and stained with eFluor 450-anti-IL-17A (clone eBio17B7; eBioscience, San Diego, CA, USA) and Alexa Flour 647-anti-IFN-γ (clone CC302; Bio-Rad) for 30 min at 4 °C. The eFluor 450-rat IgG2a kappa Isotype Control (clone eBR2a; eBioscience, San Diego, CA, USA) used for isotype control of the anti-IL-17A antibody. Singlestained samples for each antibody were used to compensate for the fluorescence spillover. The data were analyzed using FlowJo v10 software (BD Biosciences).
Intracellular survival and replication. The bacterial invasion assay was conducted as previously described with slight modification 61,62 . MDMs were infected for 2 h with M. intracellulare at an MOI of 1. Cells were washed with DPBS after centrifugation at 400×g for 5 min. Extracellular bacteria were eliminated by treatment with amikacin at a concentration of 200 µg/ml for 2 h. Following incubation, the cells were lysed with 1% Triton X-100 (Sigma-Aldrich, MO, USA). The viable intracellular bacteria were enumerated by serial dilution and plating onto Middlebrook 7H10 agar containing 0.1% casitone after vigorous vortexing for 30 s.
Statistical analysis. Statistical significance was analyzed by an unpaired t-test with Welch's correction using GraphPad Prism version 7.00 (Windows, GraphPad Software, La Jolla California USA, http:// www. graph pad. com) for comparisons between the M. intracellulare-infected and non-infected groups. Significant differences were determined at p < 0.05. Fold change is represented by the mean ratio of gene expression in M. intracellulare-infected cells/noninfected cells.

Data availability
Raw files and normalized datasets are available from the Gene Expression Omnibus (GEO) https:// www. ncbi. nlm. nih. gov/ geo/ query/ acc. cgi? acc= GSE15 8425 under the accession number GSE158425.